[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26-08-2005 to 10-02-2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

14 day RF study for 90 day study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD® (SD) BR strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

Six male and six female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. After an acclimatisation period of at least seven days, animals were selected at random and given a unique number within the range-finder by ear punching.
At the start of treatment the males weighed 128 to 155 g and the females weighed 114 to 151 g. The animals were housed in groups of three by sex in polypropylene grid-floor cages suspended over trays containing absorbent paper. Free access to mains drinking water and food (Rodent 5LF2 (Certified) Diet, BCM IPS Limited, London, UK) was allowed throughout the range-finder.
The animals were housed in a single air-conditioned room within the Safephann Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system, and printouts of the mean temperature and humidities were included in the study records. The temperature and relative humidity were controlled to remain within target ranges of 21 ± 2°C and 55 ± 15% respectively.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The substance was administered daily, for fourteen consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg/day of Corn oil.
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at Days 4, 8 and 11.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 days

Frequency of treatment:

once daily

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Two groups; each of six rats (three males and three females)

Control animals:

yes, concurrent vehicle

Details on study design:

no data

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: Yes, all animals were examined for overt signs of toxicity, ill health or behavioural change immediately before dosing, post dosing and one hour after dosing. All observations were recorded.
BODY WEIGHT: Yes, individual bodyweights were recorded on Days 1, 4, 8, 11 and 14 of the treatment period.
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: No.
HAEMATOLOGY and CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

On completion of the dosing period, all animals were killed by cervical dislocation and immediately subjected to an internal and external macroscopic examination. No tissues were retained.

Statistics:

No

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 1000 mg/kg/day showed increased salivation up to ten minutes after dosing from Day 1 onwards.

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 1000 mg/kg/day showed a slight reduction in bodyweight gain on Day 4 only. No such effects were detected for females treated with 1000 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Two males and one female treated with 1000 mg/kg/day showed pale kidneys at necropsy.

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one

EC Number:

204-846-3

EC Name:

3-methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one

Cas Number:

127-51-5

Molecular formula:

C14H22O

IUPAC Name:

3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD® (SD) BR strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Sprague-Dawley Crl:CD® (SD) BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for 6 days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 131 to 172g, the females weighed 122 to 155g, and were approximately 6 to 8 weeks old.
The animals were housed in groups of three or four by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper. The animals were allowed free access to food and water. A pelleted diet (Rodent 5LF2 (Certified) Diet, BCM IPS Limited, London, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks (B & K Universal Ltd., Hull, UK) and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
The animals were housed in a single air-conditioned room within the Safepharm Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly mean temperatures and humidities were included in the study records. The temperature and relative humidity controls were set to achieve target values of 21 ± 2°C and 55 ± 15% respectively.
The animals were randomly allocated to treatment groups using a total randomisation procedure and the group mean bodyweights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The substance was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg/day of corn oil.
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of alpha-iso-methylionone in the test material formulations was determined by gas chromatography (GC) using an external standard technique. The test material formulations were extracted with methanol to give a final, theoretical test material concentration of approximately 0.1 mg/mL. The standard and sample solutions were analysed by GC using the following conditions: GC system: Agilent Technologies 5890, incorporating autosampler and workstation, Column: DB-5 (30 m x 0.25 mm id x 0.25 μm film), Oven temperature program: initial 75°C for 0 mins rate 10°C/min final 225°C for 10 mins, Injection temperature: 250°C, Flame ionisation detector: 250°C temperature, Injection volume: 1 μL, Retention time From ~ 10.9 to 11.4 mins. The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate. The test material formulations were sampled and analysed initially and then after storage at approximately +4°C in the dark for fourteen days. The test material formulations were sampled and analysed within three days of preparation.

Duration of treatment / exposure:

90 day

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: based on the preliminary 14 day repeated dose oral (gavage) range-finder study in the rat.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes, immediately before dosing and one and five hours after dosing during the working week.
BODY WEIGHT: Yes, Individual bodyweights were recorded on Day 1 and at weekly intervals thereafter. Bodyweights were also recorded at terminal kill.
FOOD EFFICIENCY: Yes, Food consumption was recorded for each cage group at weekly intervals throughout the study.
WATER CONSUMPTION: Yes, water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.
OPHTHALMOSCOPIC EXAMINATION: Yes, The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex. Following pupil dilation with 0.5% Tropicamide solution (Alcon Laboratories (UK) Ltd., Imperial Way, Watford, Hertfordshire), detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.
HAEMATOLOGY and CLINICAL CHEMISTRY: Yes, Haematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.
URINALYSIS: No data
NEUROBEHAVIOURAL EXAMINATION: Yes, prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

Sacrifice and pathology:

On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: Adrenals, Ovaries, Brain, Spleen, Epididymides, Testes, Heart, Thymus, Kidneys, Uterus and Liver

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin: Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Epididymides, Eyes, Heart, Ileum (including Peyer's patches), Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical and mesenteric), Mammary glands, Muscle (skeletal), Oesophagus, Ovaries, Pancreas, Pituitary Bone & bone marrow (sternum), Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles, Skin (hind limb), Spinal cord (cervical, mid-thoracic and Gross lesions lumbar), Spleen, Stomach, Testes, Thymus, Thyroid/parathyroid, Tongue, Trachea, Urinary bladder, Uterus
All tissues from control and 500 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 JlID and stained with haematoxylin and eosin for subsequent microscopic examination.
Since there were indications of treatment-related changes in the liver, kidneys, thyroid gland, and bone marrow, examination was subsequently extended to include sections of these tissues from all animals in the remaining groups.

Statistics:

- Data were processed to give group mean values and standard deviations where appropriate.
- All data were summarised in tabular form. Where appropriate, quantitative data were analysed by the Provantis Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett's test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
- Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes: Chi squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater; Kruskal-Wallis one way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinically observable signs of toxicity were detected in test or control animals throughout the study period. Instances of noisy respiration and increased salivation, together with associated findings of hunched posture and tiptoe gait were evident in 500 mg/kg/day animals throughout the treatment period. Incidents of increased salivation and noisy respiration were also evident in 30 and 5 mg/kg/day animals (respectively). Observations of this nature are often reported following oral administration of an unpalatable or slightly irritant test material formulation and considered not to be an indication of systemic toxicity. One female treated with 500 and one female treated with 30 mg/kg/day showed isolated episodes of tail elevation with the latter female also showing an episode of ataxia. While the aetiology of these findings are unclear in the absence of any supporting behavioural assessments to suggest neurotoxicity, these isolated changes were considered of no toxicological importance. Isolated instances of generalised fur loss, scabs formation or generalised red-brown stained fur were evident in a number of control and treated animals throughout the dosing period. Such observations are commonly observed in laboratory maintained rats and in view of the sporadic nature of these findings were considered to be entirely incidental and unrelated to treatment. One control male showed episodes of noisy respiration whilst one control female developed hunched posture over a two day period. These findings were clearly unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse effect on bodyweight development was detected. Bodyweight gain in test animals during the treatment period was similar to that of controls. Females treated with 500 mg/kg/day showed a statistically significant reduction in bodyweight gain during Week 7 (p<0.05) together with an increase in bodyweight gain during Weeks 9 and 13 (p<0.05 and p<0.001 respectively). Mean bodyweight at termination was at least 99% of control and, in the absence of any effect on overall bodyweight gain, differences in weekly bodyweight change were considered to reflect normal biological variation and to be of no toxicological significance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no adverse effect on food consumption during the study period. Food efficiency (the ratio of bodyweight gain to dietary intake) was similar to that of controls.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles revealed no intergroup differences.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related ocular effects. The incidental findings recorded were those normally encountered in laboratory maintained rats of this age and strain.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in the haematological parameters measured. Statistical analysis revealed no significant intergroup differences.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 500 mg/kg/day showed a statistically significant increase in plasma creatinine, total protein and cholesterol (p<0.01) compared to control animals. Males from this treatment group also showed a statistically significant increase in plasma albumin (p<0.01). No such treatment-related effects were detected in animals of either sex treated with 30 or 5 mg/kg/day. Animals of either sex treated with 500 mg/kg/day showed a statistically significant reduction in aspartate aminotransferase (p<0.05 males and p<0.001 females) whilst females also showed a reduction in alkaline phosphatase (p<0.05). In the absence of any supporting data to suggest an effect of treatment these intergroup differences were considered of no toxicological importance. Males treated with 500 mg/kg/day also showed a statistically significant reduction in plasma chloride concentration (p<0.05). All individual values were within the normal range for rats of the strain and age used and the intergroup difference was considered not to be toxicologically significant. Females from all treatment groups showed a statistically significant reduction in plasma bilirubin (p<0.05). The majority of individual values were within the normal range for rats of the strain and age used and in the absence of a dose related response the intergroup differences were considered of no toxicological importance.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no treatment-related changes in the behavioural, functional performance parameters measured. There were no treatment-related changes in sensory reactivity.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 500 mg/kg/day showed a statistically significant increase in liver weight (p<0.01) both absolute and relative to terminal bodyweight. Males treated with 500 mg/kg/day also showed statistically significant increases in spleen and kidney weight (p<0.01) both absolute and relative to terminal bodyweight. Females treated with 500 mg/kg/day also showed a statistically significant increase in absolute and relative kidney weight (p<0.01). No such toxicologically significant effects were detected in animals of either sex treated with 30 or 5 mg/kg/day. Animals of either sex treated with 30 mg/kg/day showed a statistically significant reduction in absolute and relative heart weight (p<0.05). All individual values were within the normal range for rats of the strain and age used and in the absence of a dose related response the intergroup differences were considered to be of no toxicological importance. Females treated with 30 mg/kg/day showed a statistically significant increase in absolute and relative liver weight (p<0.05). In the absence of any histological correlates at this level the intergroup difference was considered not to be toxicologically significant. Males treated with 5 mg/kg/day showed a statistically significant increase in absolute and relative adrenal weight (p<0.05). In the absence of a dose-related response or any histological correlates this intergroup differences was considered not to be toxicologically significant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic abnormalities were detected.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: Hepatocyte enlargement, centrilobular or generalised, was observed in relation to treatment for 4/10 (minimal) males and 9/10 (minimal) females with 500 mg/kg/day (p< 0.05 for males; p< 0.001 for females). Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of associated degenerative changes may be interpreted as adaptive in nature.
Kidneys: A greater incidence of higher severity grades of globular accumulations of eosinophilic material were observed in the tubular epithelium of 3/10 (minimal), 5/10 (slight), 1/10 (moderate) males treated with 500 mg/kg/day (p< 0.01) or in 5/10 (minimal), 4/10 (slight) males treated with 30 mg/kg/day (p< 0.05) but not at 5 mg/kg/day. This finding may indicate the presence of hydrocarbon nephropathy, which results from the excessive accumulation of alpha-2-microglobulin in renal proximal tubular epithelial cells. Aplha-2-microglobulin is found only in the proximal tubular epithelium of adult male rats. Two males treated with 500 mg/kg/day and one male treated with 30 mg/kg/day exhibited associated higher grades of tubular basophilia.
Thyroid: A higher incidence of follicular cell hypertrophy was seen in relation to treatment for 7/10 (minimal) males treated with 500 mg/kg/day (p< 0.01).
Bone marrow: A higher incidence of lower grades of severity of adipose infiltration of the bone marrow, indicative of marrow hyperplasia, was observed in relation to treatment for 7/10 (minimal) and 3/10 (slight) males treated with 500 mg/kg/day (p< 0.01).

Histopathological findings: neoplastic:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Applicant's summary and conclusion

Conclusions:

The NOAEL in the oral 90-day repeated dose toxicity study in rats (OECD 408) is considered to be 30 mg/kg bw.

Executive summary:

In a 90-day repeated dose toxicity study, performed according to OECD Guideline 408 and in accordance with GLP, the substance was administered by oral gavage at dose levels of 500, 30 and 5 mg/kg bw/day for a period of 90 consecutive days to groups of 10 male and 10 female Sprague-Dawley rats. A control group of the same size was treated with the vehicle (corn oil) only. Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no mortalities or adverse clinical signs noted. There were no treatment-related changes in the behavioural and functional performance parameters measured. There were no treatment-related changes in sensory reactivity, bodyweight development, food consumption, water consumption, ophthalmoscopy, haematology, and no macroscopic abnormalities were detected. Animals of either sex treated with 500 mg/kg/day showed a statistically significant increase in plasma creatinine, total protein and cholesterol (p<0.01) compared to control animals. Males from this treatment group also showed a statistically significant increase in plasma albumin (p<0.01). No such effects were detected in animals of either sex treated with 30 or 5 mg/kg/day. Animals of either sex treated with 500 mg/kg/day showed a statistically significant increase in liver weight (p<0.01) both absolute and relative to terminal bodyweight. Males treated with 500 mg/kg/day also showed statistically significant increases in absolute and relative spleen weight (p<0.01) and in both sexes an increase in absolute and relative kidney weight (p<0.01) was observed. No such toxicologically significant effects were detected in animals of either sex treated with 30 or 5 mg/kg/day. Hepatocyte enlargement, centrilobular or generalised, was observed in relation to treatment for 4/10 (minimal) males and 9/10 (minimal) females treated with 500 mg/kg/day (p<0.05 for males; p<0.001 for females). Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of associated degenerative changes may be interpreted as adaptive in nature. A greater incidence of higher severity grades of globular accumulations of eosinophilic material were observed in the tubular epithelium of 3/10 (minimal), 5/10 (slight), 1/10 (moderate) males treated with 500 mg/kg/day (p<0.01) or in 5/10 (minimal), 4/10 (slight) males treated with 30 mg/kg/day (p<0.05) but not at 5 mg/kg/day. This finding is consistent with the presence of hydrocarbon nephropathy, which results from the excessive accumulation of alpha2-microglobulin in renal proximal tubular epithelial cells. This is a well documented effect, peculiar to the male rat, which occurs in response to treatment with certain hydrocarbons. Female rats and other species do not develop "hydrocarbon nephropathy" and for this reason the effect may not be indicative of hazard to human health. Two males treated with 500 mg/kg/day and one male treated with 30 mg/kg/day exhibited associated higher grades of tubular basophilia. A higher incidence of follicular cell hypertrophy was seen in relation to treatment for 7/10 (minimal) males treated with 500 mg/kg/day (p<0.01). A higher incidence of lower grades of severity of adipose infiltration of the bone marrow, indicative of marrow hyperplasia, was observed in relation to treatment for 7/10 (minimal) and 3/1 (slight) males treated with 500 mg/kg/day (p<0.01).

The NOAEL for systemic toxicity has been set at 30 mg/kg bw/day, based on increases in plasma levels of creatinine, total protein and cholesterol (indicators of liver effects), increases in absolute and relative weights of the kidneys and spleen, and microscopic changes in thyroid and bone marrow at the next dose level.